Heterotandem bicyclic peptide complex

ABSTRACT

The present invention relates to a heterotandem bicyclic peptide complex which comprises a first peptide ligand, which binds to EphA2, conjugated via a linker to two second peptide ligands, which bind to CD137. The invention also relates to the use of said heterotandem bicyclic peptide complex in preventing, suppressing or treating cancer.

FIELD OF THE INVENTION

The present invention relates to a heterotandem bicyclic peptide complex which comprises a first peptide ligand, which binds to EphA2, conjugated via a linker to two second peptide ligands, which bind to CD137. The invention also relates to the use of said heterotandem bicyclic peptide complex in preventing, suppressing or treating cancer.

BACKGROUND OF THE INVENTION

Cyclic peptides can bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.

Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem).

Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) are disclosed in WO 2019/122860 and WO 2019/122863.

Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to EphA2 and which has         the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1NaI]WTC_(iii)         (SEQ ID NO: 1; BCY13118); conjugated via an         N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to     -   (b) two second peptide ligands which bind to CD137 both of which         have the sequence         Ac-C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ         ID NO: 2; BCY8928);         wherein each of said peptide ligands comprise a polypeptide         comprising three reactive cysteine groups (C_(i), C_(ii) and         C_(iii)), separated by two loop sequences, and a molecular         scaffold which is         1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)         and which forms covalent bonds with the reactive cysteine groups         of the polypeptide such that two polypeptide loops are formed on         the molecular scaffold;         wherein Ac represents acetyl, HArg represents homoarginine, HyP         represents trans-4-hydroxy-L-proline, d1NaI represents         D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA         represents 4-pentynoic acid and Nle represents norleucine.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a heterotandem bicyclic peptide complex as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Analysis of the EphA2/CD137 heterotandem bicyclic peptide complex BCY13272 in the Promega CD137 luciferase reporter assay in the presence of EphA2 expressing A549, PC-3 and HT29 cells (n=3). BCY13626 is a heterotandem bicyclic peptide complex similar to BCY13272 but comprises D-amino acids and does not bind to EphA2 or CD137.

FIG. 2: Plasma concentration versus time plot of BCY13272 from a 5.5 mg/kg IV dose in CD1 mice (n=3), a 3.6 mg/kg IV infusion (15 min) in SD rats (n=3) and a 8.9 mg/kg IV infusion (15 min) in cynomolgus monkeys (n=2). The pharmacokinetic profile of BCY13272 has a terminal half-life of 2.9 hours in CD-1 mice, 2.5 hours in SD Rats and 8.9 hours in cyno.

FIG. 3: Anti-tumor activity of BCY13272 in a syngeneic MC38 tumor model. (A) MC38 tumor volumes during and after BCY13272 treatment. Number of complete responder (CR) mice on D28 (and that remain CRs on D62) are indicated in parentheses. BIW: twice weekly dosing; IV: intravenous administration. (B) Tumor growth curves of complete responder animals to BCY13272 and naïve age-matched control animals after MC38 tumor cell implantation. CR: complete responder.

FIG. 4: BCY13272 induces IFN-γ cytokine secretion in a (A) PBMC/MC38 and a (B) PBMC/HT29 co-culture assay. BCY12762 is a heterotandem bicyclic peptide complex that binds to EphA2 but does not bind to CD137. BCY13692 is a heterotandem bicycle peptide complex that binds to CD137 but does not bind to EphA2. (C) Plot of EC50 (nM) values of BCY13272 induced IL-2 and IFN-y secretion in PBMC coculture assay with MC38 (mouse) cell line with 5 PBMC donors and HT1080 (human) cell line with 4 PBMC donors.

FIG. 5: Surface plasmon resonance (SPR) binding of BCY13272 to immobilized (A) EphA2 and (B) CD137.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to EphA2 and which has         the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1NaI]WTC_(iii)         (SEQ ID NO: 1; BCY13118); conjugated via an         N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to     -   (b) two second peptide ligands which bind to CD137 both of which         have the sequence         Ac-C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ         ID NO: 2; BCY8928); wherein each of said peptide ligands         comprise a polypeptide comprising three reactive cysteine groups         (C_(i), C_(ii) and C_(iii)), separated by two loop sequences,         and a molecular scaffold which is         1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)         and which forms covalent bonds with the reactive cysteine groups         of the polypeptide such that two polypeptide loops are formed on         the molecular scaffold;         wherein Ac represents acetyl, HArg represents homoarginine, HyP         represents trans-4-hydroxy-L-proline, d1NaI represents         D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA         represents 4-pentynoic acid and Nle represents norleucine.

References herein to a N-(acid-PEG₃)-N-bis(PEG₃-azide) linker include:

In one embodiment, the heterotandem bicyclic peptide complex is BCY13272:

Full details of BCY13272 are shown in Table A below:

TABLE A Composition of BCY13272 Complex EphA2 Attachment CD137 Attachment No. BCY No. Point Linker BCY No. Point BCY13272 BCY13118 N-terminus N-(acid-PEG₃)- BCY8928, dLys (PYA)4 N-bis(PEG₃-azide) BCY8928

Data is presented here in FIG. 3 which demonstrates that BCY13272 leads to a significant anti tumor effect in a MC38 tumor model in mice.

Reference herein is made to certain analogues (i.e. modified derivatives) and metabolites of BCY13272, each of which form additional aspects of the invention and are summarised in Table B below:

TABLE B Composition of labelled analoques and potential metabolites of BCY13272 Complex EphA2 Attachment CD137 Attachment No. BCY No. Point Linker BCY No. Point Modifier BCY14414 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 N/A terminus bis(PEG₃-azide) BCY13389 dLys(PYA)4 BCY14417 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 Peg12-Biotin terminus bis(PEG₃-azide) BCY13389 dLys(PYA)4 BCY14418 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 Alexa terminus bis(PEG₃-azide) BCY13389 dLys(PYA)4 Fluor ® 488 BCY15217 BCY13118 N- N-(acid-PEG₃)-N- BCY14601 dLys(PYA)4 N/A terminus bis(PEG₃-azide) BCY14601 dLys(PYA)4 BCY15218 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 N/A terminus bis(PEG₃-azide) BCY14601 dLys(PYA)4 wherein BCY14601 represents a bicyclic peptide ligand having the sequence of C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ ID NO: 3) with TATA as a molecular scaffold; and wherein BCY13389 represents a bicyclic peptide ligand having the sequence of [Ac]C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-K (SEQ ID NO: 4) with TATA as a molecular scaffold.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th) ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within compounds of the invention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within SEQ ID NO: 1 is referred to as below:

(SEQ ID NO: 1) C_(i)HyP₁-L₂-V₃-N₄-P₅-L₆-C_(ii)-L₇-E₈-P₉-d1Nal₁₀-W₁₁-T₁₂-C_(iii).

For the purpose of this description, the bicyclic peptides are cyclised with 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and yield a tri-substituted structure. Cyclisation with TATA occurs on C_(i), C_(ii) and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail would be denoted as:

(SEQ ID NO: X) βAla-Sar10-A.

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa). For the avoidance of doubt, references to amino acids either as their full name or as their amino acid single or three letter codes are intended to be represented herein as L-amino acids unless otherwise stated. If such an amino acid is intended to be represented as a D-amino acid then the amino acid will be prefaced with a lower case d within square parentheses, for example [dA], [dD], [dE], [dK], [d1NaI], [dNle], etc.

Advantages of the Peptide Ligands

Certain heterotandem bicyclic peptide complexes of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

-   -   Species cross-reactivity. This is a typical requirement for         preclinical pharmacodynamics and pharmacokinetic evaluation;     -   Protease stability. Heterotandem bicyclic peptide complexes         should ideally demonstrate stability to plasma proteases,         epithelial (“membrane-anchored”) proteases, gastric and         intestinal proteases, lung surface proteases, intracellular         proteases and the like. Protease stability should be maintained         between different species such that a heterotandem bicyclic         peptide lead candidate can be developed in animal models as well         as administered with confidence to humans;     -   Desirable solubility profile. This is a function of the         proportion of charged and hydrophilic versus hydrophobic         residues and intra/inter-molecular H-bonding, which is important         for formulation and absorption purposes;     -   Selectivity. Certain heterotandem bicyclic peptide complexes of         the invention demonstrate good selectivity over other targets;     -   An optimal plasma half-life in the circulation. Depending upon         the clinical indication and treatment regimen, it may be         required to develop a heterotandem bicyclic peptide complex for         short exposure in an acute illness management setting, or         develop a heterotandem bicyclic peptide complex with enhanced         retention in the circulation, and is therefore optimal for the         management of more chronic disease states. Other factors driving         the desirable plasma half-life are requirements of sustained         exposure for maximal therapeutic efficiency versus the         accompanying toxicology due to sustained exposure of the agent.     -   Crucially, data is presented herein where the heterotandem         bicyclic peptide complex of the invention demonstrates         anti-tumor efficacy when dosed at a frequency that does not         maintain plasma concentrations above the in vitro EC50 of the         compound. This is in contrast to larger recombinant biologic         (i.e. antibody based) approaches to CD137 agonism or bispecific         CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936         (2017), Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12         (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889         (2019)). Without being bound by theory, the reason for this         observation is thought to be due to the fact that heterotandem         bicycle complexes have relatively low molecular weight         (typically <15 kDa), they are fully synthetic and they are tumor         targeted agonists of CD137. As such, they have relatively short         plasma half lives but good tumor penetrance and retention. Data         is presented herein which fully supports these advantages. For         example, anti-tumor efficacy in syngeneic rodent models in mice         with humanized CD137 is demonstrated either daily or every         3^(rd) day. In addition, intraperitoneal pharmacokinetic data         shows that the plasma half life is <3 hours, which would predict         that the circulating concentration of the complex would         consistently drop below the in vitro EC₅₀ between doses.         Furthermore, tumor pharmacokinetic data shows that levels of         heterotandem bicycle complex in tumor tissue may be higher and         more sustained as compared to plasma levels.     -   It will be appreciated that this observation forms an important         further aspect of the invention. Thus, according to a further         aspect of the invention, there is provided a method of treating         cancer which comprises administration of a heterotandem bicyclic         peptide complex as defined herein at a dosage frequency which         does not sustain plasma concentrations of said complex above the         in vitro EC50 of said complex.     -   Immune Memory. Coupling the cancer cell binding bicyclic peptide         ligand with the immune cell binding bicyclic peptide ligand         provides the synergistic advantage of immune memory. The         heterotandem bicyclic peptide complex of the invention is         believed not only to eradicate tumors but upon readministration         of the tumorigenic agent, none of the inoculated complete         responder mice developed tumors. This indicates that treatment         with the heterotandem bicyclic peptide complex of the invention         has induced immunogenic memory in the complete responder mice.         This has a significant clinical advantage in order to prevent         recurrence of said tumor once it has been initially controlled         and eradicated.

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide is bound to the scaffold. In the present case, the peptides comprise at least three reactive groups selected from cysteine, 3-mercaptopropionic acid and/or cysteamine and form at least two loops on the scaffold.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO⁻), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁻) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄₊.

Where the compounds of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.

In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (the group referred to herein as C_(i)) is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group (the group referred to herein as C_(iii)) is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca-disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (C_(i)) and/or the C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise β-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic         effect and lead to lower off rates, such that higher affinities         are achieved;     -   Incorporating charged groups that exploit long-range ionic         interactions, leading to faster on rates and to higher         affinities (see for example Schreiber et al, Rapid,         electrostatically assisted association of proteins (1996),         Nature Struct. Biol. 3, 427-31); and     -   Incorporating additional constraint into the peptide, by for         example constraining side chains of amino acids correctly such         that loss in entropy is minimal upon target binding,         constraining the torsional angles of the backbone such that loss         in entropy is minimal upon target binding and introducing         additional cyclisations in the molecule for identical reasons.         (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,         (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem         (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T), carbon, such as 11C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur, such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga, yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as ²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the Nectin-4 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Synthesis

The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides or conjugates selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide/conjugate made by chemical synthesis.

Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptides, forming a disulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

Pharmaceutical Compositions According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringers dextrose, dextrose and sodium chloride and lactated Ringers. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringers dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a “therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing a peptide ligand according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

Therapeutic Uses

According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer.

Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumors of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenousleukemia [AML], chronic myelogenousleukemia [CML], chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocyticleukemia); tumors of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcomaprotuberans; tumors of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (for example pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary carcinoma of the thyroid); ocular and adnexal tumors (for example retinoblastoma); germ cell and trophoblastic tumors (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumors (for example medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).

In a further embodiment, the cancer is selected from a hematopoietic malignancy such as selected from: non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

References herein to the term “prevention” involves administration of the protective composition prior to the induction of the disease. “Suppression” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.

“Treatment” involves administration of the protective composition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.

The invention is further described below with reference to the following examples.

EXAMPLES

In general, the heterotandem bicyclic peptide complex of the invention may be prepared in accordance with the following general method:

All solvents are degassed and purged with N₂ 3 times. A solution of BP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF is mixed for 5 minutes, then Bicycle1 (1.2 eq.) is added. The reaction mixture is stirred at 40° C. for 16 hr. The reaction mixture is then concentrated under reduced pressure to remove solvent and purified by prep-HPLC to give intermediate 2.

A mixture of intermediate 2 (1.0 eq) and Bicycle2 (2.0 eq) are dissolved in t-BuOH/H₂O (1:1), and then CuSO₄ (1.0 eq), VcNa (4.0 eq), and THPTA (2.0 eq) are added. Finally, 0.2 M NH₄HCO₃ is added to adjust pH to 8. The reaction mixture is stirred at 40° C. for 16 hr under N₂ atmosphere. The reaction mixture was directly purified by prep-HPLC.

More detailed experimental for the heterotandem bicyclic peptide complex of the invention is provided herein below:

Example 1: Synthesis of BCY13272

Procedure for Preparation of BCY14964

A mixture of BP-23825 (155.5 mg, 249.40 μmol, 1.2 eq), and HATU (95.0 mg, 249.92 μmol, 1.2 eq) was dissolved in NMP (1.0 mL), then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg, 499.83 μmol, 87.0 μL, 2.4 eq), and then the solution was allowed to stir at 25° C. for 5 min. BCY13118 (500.0 mg, 207.83 μmol, 1.0 eq) was dissolved in NMP (5.0 mL), and then added to the reaction solution, the pH of the resulting solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 45 min. LC-MS showed BCY13118 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by preparative-HPLC to give BCY14964 (1.35 g, 403.46 μmol, 64.7% yield, 90% purity) as a white solid. Calculated MW: 3011.53, observed m/z: 1506.8 ([M+2H]²⁺), 1005.0 ([M+3H]³⁺).

Procedure for Preparation of BCY13272

A mixture of BCY8928 (644.0 mg, 290.55 μmol, 2.5 eq), THPTA (50.5 mg, 116.22 μmol, 1.0 eq), CuSO₄ (0.4 M, 145.0 μL, 0.5 eq) and sodium ascorbate (82.0 mg, 464.89 μmol, 4.0 eq) were dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 6.0 mL). The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃), and then the solution was stirred at 25° C. for 3 min. BCY14964 (350.0 mg, 116.22 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 11.0 mL), and then dropped into the stirred solution. All solvents here were pre-degassed and purged with N₂. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 6 hr under N₂ atmosphere. LC-MS showed one main product peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY13272 (1.75 g, 235.01 μmol, 67.40% yield, 94% purity) was obtained as a white solid. Calculated MW: 7446.64, observed m/z: 1242.0 ([M+6H]⁶⁺), 1491.0 ([M+5H]⁵⁺).

Example 2: Synthesis of BCY14414

Procedure for Preparation of BCY14798

A mixture of BCY14964 (55.0 mg, 18.26 μmol, 1.0 eq), BCY8928 (32.4 mg, 14.61 μmol, 0.8 eq), and THPTA (39.8 mg, 91.32 μmol, 5.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂), and then CuSO₄ (0.4 M, 23.0 μL, 0.5 eq) and sodium ascorbate (72.0 mg, 365.27 μmol, 20.0 eq) were added under N₂. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃), and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 1.5 h under N₂ atmosphere. LC-MS showed BCY14964 remained, compound BCY8928 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY14798 (51 mg, 9.17 μmol, 33.37% yield, 94% purity) was obtained as a white solid. Calculated MW: 5229.07, observed m/z: 1308.3 ([M+4H]⁴⁺), 1046.7 ([M+5H]⁵⁺).

Procedure for Preparation of BCY14414

A mixture of BCY14798 (21.0 mg, 4.02 μmol, 1.0 eq), BCY13389 (10.0 mg, 4.42 μmol, 1.1 eq), and THPTA (1.8 mg, 4.02 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂), and then CuSO₄ (0.4 M, 5.0 μL, 0.5 eq) and sodium ascorbate (2.8 mg, 16.06 μmol, 4.0 eq) were added under N₂. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃) and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showed BCY14798 was consumed completely, some BCY13389 remained and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative and BCY14414 (20 mg, 2.40 μmol, 59.73% yield, 90.9% purity) was obtained as a white solid. Calculated MW: 7503.74, observed m/z: 1251.5 ([M+5H]⁵⁺), 1072.9 ([M+7H]⁷⁺).

Example 3: Synthesis of BCY14417

Procedure for Preparation of BCY14417

A mixture of BCY14414 (13.0 mg, 1.73 μmol, 1.0 eq) and biotin-PEG₁₂-NHS ester (CAS 365441-71-0, 4.2 mg, 4.50 μmol, 2.6 eq) was dissolved in DMF (0.5 mL). The pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MS showed BCY14414 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC and BCY14417 (9.0 mg, 1.07 μmol, 80.49% yield, 90.8% purity) was obtained as a white solid. Calculated MW: 8329.74, observed m/z: 1389.6 ([M+6H]⁶⁺), 1191.9 ([M+7H]⁷⁺).

Example 4: Synthesis of BCY14418

Procedure for Preparation of BCY14418

A mixture of BCY14414 (5.6 mg, 0.75 μmol, 1.0 eq) and Alexa Fluor® 488 (0.9 mg, 1.49 μmol, 2.0 eq) was dissolved in DMF (0.3 mL). Then pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 1.0 hr. LC-MS showed BCY14414 was consumed completely, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue.

The crude product was purified by preparative HPLC, and BCY14418 (2.3 mg, 0.25 μmol, 32.89% yield, 85.6% purity) was obtained as a red solid. Calculated MW: 8020.19, observed m/z: 1337.2 ([M+6H]⁶⁺).

Example 5: Synthesis of BCY15217

Procedure for Preparation of BCY15217

A mixture of BCY14964 (20.0 mg, 6.64 μmol, 1.0 eq), BCY14601 (30.5 mg, 13.95 μmol, 2.1 eq), and THPTA (2.9 mg, 6.64 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂), and then CuSO₄ (0.4 M, 16.6 μL, 1.0 eq) and sodium ascorbate (4.7 mg, 26.56 μmol, 4.0 eq) were added under N₂. The pH of this solution was adjusted to 8, and the solution turned to light yellow. The reaction mixture was stirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showed BCY14964 remained, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY15217 (19.7 mg, 2.41 μmol, 36.26% yield, 96.2% purity) was obtained as a white solid. Calculated MW: 7362.5, observed m/z: 1473.5 ([M+5H]⁵⁺), 1228.2 ([M+6H]⁶⁺), 1052.8 ([M+7H]⁷⁺).

Example 6: Synthesis of BCY15218

Procedure for Preparation of BCY15218

A mixture of BCY14798 (30.0 mg, 5.74 μmol, 1.0 eq), BCY14601 (15.0 mg, 6.88 μmol, 1.2 eq), and THPTA (2.5 mg, 5.74 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂), and then CuSO₄ (0.4 M, 14.0 μL, 1.0 eq) and sodium ascorbate (4.0 mg, 22.95 μmol, 4.0 eq) were added under N₂. The pH of this solution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃), and the solution turned light yellow. The reaction mixture was stirred at 25° C. for 2 h under N₂ atmosphere. LC-MS showed BCY14798 was consumed completely, BCY14601 remained, and one main peak with desired m/z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC, and BCY15218 (22 mg, 2.67 μmol, 46.61% yield, 95.0% purity) was obtained as a white solid. Calculated MW: 7404.6, observed m/z: 1234.8 ([M+6H]⁶⁺).

Analytical Data

The heterotandem bicyclic peptide complex of the invention was analysed using mass spectrometry and HPLC. HPLC setup was as follows:

-   -   Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA in ACN     -   Flow: 1.0 ml/min     -   Column: Kintex 1.7 um C18 100 A 2.1 mm*150 mm     -   Instrument: Agilent UPLC 1290

Gradients used are 30-60% B over 10 minutes and the data was generated as follows:

HPLC Complex Analytical Data- Retention ID Mass Spectrometry Time (min) BCY13272 Calculated MW: 7102.28, observed m/z: 7.07 1776.4 [M + 4H]4+, 1421.3 [M + 5H]+

Biological Data

1. CD137 Reporter Assay Co-Culture with Tumor Cells

Culture medium, referred to as R1 media, is prepared by adding 1% FBS to RPMI-1640 (component of Promega kit CS196005). Serial dilutions of test articles in R1 are prepared in a sterile 96 well-plate. Add 25 μL per well of test articles or R1 (as a background control) to designated wells in a white cell culture plate. Tumor cells* are harvested and resuspended at a concentration of 400,000 cells/mL in R1 media. Twenty five (25) μL/well of tumor cells are added to the white cell culture plate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in the water bath and then added to 5 ml pre-warmed R1 media. Twenty five (25) μL/well of Jurkat cells are then added to the white cell culture plate. Incubate the cells and test articles for 6 h at 37° C., 5% CO₂. At the end of 6 h, add 75 μL/well Bio-Glo™ reagent (Promega) and incubate for 10 min before reading luminescence in a plate reader (Clariostar, BMG). The fold change relative to cells alone (Jurkat cells+Cell line used in co-culture) is calculated and plotted in GraphPad Prism as log(agonist) vs response to determine EC₅₀ (nM) and Fold Induction over background (Max).

The tumor cell types used in co-culture for EphA2 are A549, PC-3 and HT29.

Data presented in FIG. 1 shows that the EphA2/CD137 heterotandem BCY13272 induces strong CD137 activation in a CD137 reporter assay in the presence of an EphA2 expressing cell line (A549) while a non-binding control molecule (BCY13626) shows no activation of CD137.

Data presented in FIG. 1 and in Table 1 below shows that BCY13272 induces strong CD137 activation in a CD137 reporter assay. The activation is dependent on the binding of the heterotandem to both CD137 and EphA2 as shown by the absence of activity of a non-binding control (BCY13626) which does not engage EphA2 or CD137.

A summary of the EC₅₀ (nM) and Fold Induction induced by BCY13272 in a CD137 reporter assay in co-culture with an EphA2 expressing tumor cell line is reported in Table 1 below:

TABLE 1 Activity of EphA2/CD137 heterotandem bicyclic peptide complexes in a CD137 reporter assay Geo mean Complex EphA2 EC50 EC50/cell ID cell line (nM) Emax line BCY13272 PC-3 0.245 44.5 0.117 0.0805 44.2 0.0898 53 A549 0.1468 25.7 0.127 0.107 23.6 0.132 30.2 HT-29 0.567 36.5 0.279 0.187 26 0.205 36.4

2. Pharmacokinetics of Heterotandem Complex BCY13272 in SD Rats

Male SD Rats were dosed with heterotandem complex BCY13272 formulated in 25 mM Histidine HCl, 10% sucrose pH 7 by IV bolus or IV infusion (15 minutes). Serial bleeding (about 80 μL blood/time point) was performed via submandibular or saphenous vein at each time point. All blood samples were immediately transferred into prechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4° C., 3000 g. The precipitant including internal standard was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10 minutes. The supernatant was transferred into pre-labeled polypropylene microcentrifuge tubes, and then quick-frozen over dry ice. The samples were stored at 70° C. or below as needed until analysis. 7.5 μL of the supernatant samples were directly injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T1/2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported. The pharmacokinetic parameters from the experiment are as shown in Table 2:

TABLE 2 Pharmacokinetic Parameters in SD Rats Dosing Vdss Clp Compound Route T1/2(h) (L/kg) (ml/min/kg) BCY13272 IV Inf 2.5 1.0 7.4

3. Pharmacokinetics of Heterotandem Complex BCY13272 in Cynomolgus Monkey

Non-naïve Cynomolgus Monkeys were dosed via intravenous infusion (15 or 30 min) into the cephalic vein with 1 mg/kg of heterotandem complex BCY13272 formulated in 25 mM Histidine HCl, 10% sucrose pH 7. Serial bleeding (about 1.2 ml blood/time point) was performed from a peripheral vessel from restrained, non-sedated animals at each time point into a commercially available tube containing potassium (K2) EDTA*2H₂O (0.85-1.15 mg) on wet ice and processed for plasma. Samples were centrifuged (3,000×g for 10 minutes at 2 to 8° C.) immediately after collection. 0.1 mL plasma was transferred into labelled polypropylene micro-centrifuge tubes. 5-fold of the precipitant including internal standard 100 ng/mL Labetalol & 100 ng/mL dexamethasone & 100 ng/mL tolbutamide & 100 ng/mL Verapamil & 100 ng/mL Glyburide & 100 ng/mL Celecoxib in MeOH was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm for 10 minutes at 2 to 8° C. Samples of supernatant were transferred into the pre-labeled polypropylene microcentrifuge tubes, and frozen over dry ice. The samples were stored at −60° C. or below until LC-MS/MS analysis. An aliquot of 40 μL calibration standard, quality control, single blank and double blank samples were added to the 1.5 mL tube. Each sample (except the double blank) was quenched with 200 μL IS1 respectively (double blank sample was quenched with 200 μL MeOH with 0.5% tritonX-100), and then the mixture was vortex-mixed well (at least 15 s) with vortexer and centrifuged for 15 min at 12000 g, 4° C. A 10 μL supernatant was injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T1/2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported. The pharmacokinetic parameters for BCY13272 are as shown in Table 3.

TABLE 3 Pharmacokinetic Parameters in cynomolgous monkey T_(1/2) Clp Vdss Compound Route (h) (ml/min/kg) (L/kg) BCY13272 IV infusion 8.9 4.1 0.82 (15 min)

FIG. 2 shows the plasma concentration vs time curve of BCY13272 from a 3.6 mg/kg IV infusion (15 min) in SD Rat (n=3) and a 9.2 mg/kg IV infusion (15 min) in cynomolgus monkey (n=3). BCY13272 has a volume of distribution at steady state (Vdss) of 1.0 L/kg and a clearance of 7.5 mL/min/kg in rats which results in a terminal half life of 2.9 h. BCY13272 has a volume of distribution at steady state (Vdss) of 0.82 L/kg and a clearance of 4.1 mL/min/kg in cyno which results in a terminal half life of 8.9 h.

4. Pharmacokinetics of Heterotandem Complex BCY13272 in CD1 Mice

6 Male CD-1 mice were dosed with 15 mg/kg of heterotandem complex BCY13272 formulated in 25 mM Histidine HCl, 10% sucrose pH 7 via intraperitoneal or intravenous administration. Serial bleeding (about 80 μL blood/time point) was performed via submandibular or saphenous vein at each time point. All blood samples were immediately transferred into prechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4° C., 3000 g. The precipitant including internal standard was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10 minutes. The supernatant was transferred into pre-labeled polypropylene microcentrifuge tubes, and then quick-frozen over dry ice. The samples were stored at 70° C. or below as needed until analysis. 7.5 μL of the supernatant samples were directly injected for LC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of analyte. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C0, CI, Vdss, T1/2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus time profile were reported.

FIG. 2 shows the plasma concentration vs time curve of BCY13272 from a 5.5 mg/kg IV dose in CD1 mice (n=3); the volume of distribution (Vdss) of BCY13272 is 1.1 L/kg with a Clearance of 7.5 mL/min/kg which results in terminal plasma half life of 2.9 h.

5. EphA2/CD137 Heterotandem Bicyclic Peptide Complex BCY13272 Induces IFN-γ Cytokine Secretion in an MC38 Co-Culture Assay

MC38 and HT1080 cell lines were cultured according to recommended protocols. Frozen PBMCs from healthy human donors were thawed and washed once in room temperature PBS with benzonase, and then resuspended in RPMI supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 1× Penicillin/Streptomycin, 10 mM HEPES, and 2 mM L-Glutamine (herein referred to as R10 medium). 100 μl of PBMCs (1,000,000 PBMCs/ml) and 100 μl of tumor cells (100,000 tumor cells/ml) (Effector: Target cell ratio (E:T) 10:1) were plated in each well of a 96 well flat bottom plate for the co-culture assay. 100 ng/ml of soluble anti-CD3 mAb (clone OKT3) was added to the culture on day 0 to stimulate human PBMCs. Test, control compounds, or vehicle controls were diluted in R10 media and 50 μL was added to respective wells to bring the final volume per well to 250 μL. Plates were covered with a breathable film and incubated in a humidified chamber at 37° C. with 5% CO₂ for two days. Supernatants were collected 24 and 48 hours after stimulation, and human IFN-γ was detected by Luminex. Briefly, the standards and samples were added to a black 96 well plate. Microparticle cocktail (provided in Luminex kit, R&D Systems) was added and shaken for 2 hours at room temperature. The plate was washed 3 times using a magnetic holder. Biotin cocktail was then added to the plate and shaken for 1 hour at RT. The plate was washed 3 times using a magnetic holder. Streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plates were washed 3 times using a magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT, and read using the Luminex 2000. Raw data were analyzed using built-in Luminex software to generate standard curves and interpolate protein concentrations, all other data analyses and graphing were performed using Excel and Prism software. Data represents one study with three independent donor PBMCs tested in experimental duplicates.

Data presented in FIG. 2 and in Table 4 below shows that BCY13272 induces strong CD137 activation as evidenced by IFN-y and IL-2 secretion upon CD3 stimulation. The activation is dependent on the binding of the heterotandem to both CD137 and EphA2 as evidenced by the lack of activity of the non-binding controls BCY12762 and BCY13692 where the CD137 and EphA2 binders respectively comprise all D-amino acid which result in a non-binding analog.

TABLE 4 EC50 of IL-2 cytokine secretion induced by EphA2/CD137 heterotandem bicyclic complex BCY13272 in human PBMC-MC38/HT-1080 co-culture assay Complex ID Cell line EC₅₀ (nM) N = BCY13272 MC38 0.79 ± 0.24 5 BCY13272 HT-1080 0.55 ± 0.47 4

6. Anti-Tumor Activity of BCY13272 in a Syngeneic MC38 Tumor Model

6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with 1×10⁶ MC38 cells. Mice were randomized into treatment groups (n=6/cohort) when average tumor volumes reached around 80 mm³ and were treated with vehicle (25 mM histidine, 10% sucrose, pH7) intravenously (IV), 8 mg/kg BCY13272, 0.9 mg/kg BCY13272 and 0.1 mg/kg BCY13272 IV. All treatments were given twice a week (BIW) for 6 doses in total. Tumor growth was monitored until Day 28 from treatment initiation. Complete responder animals (n=7) were followed until day 62 after treatment initiation and re-challenged with an implantation of 2×10⁶ MC38 tumor cells and tumor growth was monitored for 28 days. In parallel, naïve age-matched control huCD137 C57Bl/6 mice (n=5) were implanted with 2×10⁶ MC38 tumor cells monitored for 28 days. The results of this experiment may be seen in FIG. 3 where it can be seen that BCY13272 leads to significant anti-tumor activity with complete responses observed at 0.9 (2 out of 6 complete responders) and 8 mg/kg (5 out of 6 complete responders) dose levels (FIG. 3A). Unlike in naïve age-matched control huCD137 C57Bl/6 mice (tumor take rate 100%), no tumor regrowth was observed in BCY13272 complete responder animals (FIG. 3B). These data indicate that BCY13272 has significant anti-tumor activity and that the BCY13272 treatment can lead into immunogenic memory in the complete responder animals.

7. Binding of BCY13272 to EphA2 and CD137 as Measured by SPR

(a) CD137

Biacore experiments were performed to determine k_(a) (M⁻¹ s⁻¹), k_(d) (s⁻¹), K_(D) (nM) values of heterotandem peptides binding to human CD137 protein. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer's suggested protocol. The protein was desalted to remove uncoupled biotin using spin columns into PBS.

For analysis of peptide binding, a Biacore T200 or a Biacore 3000 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl of onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) and biotinylated CD137 captured to a level of 270-1500 RU. Buffer was changed to PBS/0.05% Tween 20 and a dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The top peptide concentration was 500 nM with 6 further 2-fold or 3-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/min with 60 seconds association and 900 seconds dissociation. After each cycle a regeneration step (10 μl of 10 mM glycine pH 2) was employed. Data were corrected for DMSO excluded volume effects as needed. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate.

(b) EphA2

Biacore experiments were performed to determine k_(a) (M⁻¹ s⁻¹), k_(d) (s⁻¹), K_(D) (nM) values of BCY13272 binding to human EphA2 protein.

EphA2 were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 with a 3× molar excess of biotin over protein. The degree of labelling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture into PBS. For analysis of peptide binding, a Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N(1:1). Buffer was changed to PBS/0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500-1500 RU using a dilution of protein to 0.2 μM in buffer. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5% with a top peptide concentration was 50 or 100 nM and 6 further 2-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/min with 60 seconds association and 900-1200 seconds dissociation. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate.

FIG. 5A shows the sensorgram which demonstrates that BCY13272 binds to EphA2 (human) with an affinity of 2.0 nM. FIG. 5B shows the sensorgram that BCY13272 binds to CD137 (human) with high affinity. Due to the presence of 2 CD137 binding bicycles in

BCY13272, the off rate from immobilized CD137 protein is very slow and the reported K_(D) may be an overestimation (FIG. 4B). 

1. A heterotandem bicyclic peptide complex comprising: (a) a first peptide ligand which binds to EphA2 and which has the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1NaI]WTC_(iii) (SEQ ID NO: 1); conjugated via an N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to (b) two second peptide ligands which bind to CD137 both of which have the sequence Ac-C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ ID NO: 2); or a pharmaceutically acceptable salt thereof, wherein each of said peptide ligands comprise a polypeptide comprising three reactive cysteine groups (C_(i), C_(ii) and C_(iii)), separated by two loop sequences, and a molecular scaffold which is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and which forms covalent bonds with the reactive cysteine groups of the polypeptide such that two polypeptide loops are formed on the molecular scaffold; wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, 1NaI represents 1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid and Nle represents norleucine.
 2. The heterotandem bicyclic peptide complex according to claim 1 which is:

or a pharmaceutically acceptable salt thereof.
 3. The heterotandem bicyclic peptide complex as defined in claim 1, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt.
 4. A pharmaceutical composition which comprises the heterotandem bicyclic peptide complex of claim 1, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable excipients.
 5. A method of preventing, suppressing or treating cancer, comprising administering the heterotandem bicyclic peptide complex as defined in claim 1, or a pharmaceutically acceptable salt thereof.
 6. A method of treating cancer which comprises administration of the heterotandem bicyclic peptide complex as defined in claim 1 or a pharmaceutically acceptable salt thereof, at a dosage frequency which does not sustain plasma concentrations of said complex above the in vitro EC50 of said complex.
 7. The method of claim 5, wherein the cancer is lung cancer, colon cancer, bladder cancer, or breast cancer.
 8. The method of claim 6, wherein the cancer is lung cancer, colon cancer, bladder cancer, or breast cancer.
 9. The method of claim 6, wherein the dosage frequency is twice per week.
 10. The method of claim 6, wherein the heterotandem bicyclic peptide complex, or a pharmaceutically acceptable salt thereof, is administered via an intravenous administration.
 11. A heterotandem bicyclic peptide complex which is:

or a pharmaceutically acceptable salt thereof.
 12. The heterotandem bicyclic peptide complex of claim 11, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt.
 13. A pharmaceutical composition comprising the heterotandem bicyclic peptide complex of claim 11, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable excipients.
 14. A method of preventing, suppressing or treating cancer, comprising administering the heterotandem bicyclic peptide complex of claim 11, or a pharmaceutically acceptable salt thereof.
 15. The method of claim 14, wherein the cancer is lung cancer, colon cancer, bladder cancer, or breast cancer.
 16. A method of treating cancer which comprises administration of the heterotandem bicyclic peptide complex of claim 11, or a pharmaceutically acceptable salt thereof, at a dosage frequency which does not sustain plasma concentrations of said complex above the in vitro EC₅₀ of said complex.
 17. The method of claim 16, wherein the cancer is lung cancer, colon cancer, bladder cancer, or breast cancer.
 18. The method of claim 16, wherein the dosage frequency is twice per week.
 19. The method of claim 16, wherein the heterotandem bicyclic peptide complex, or a pharmaceutically acceptable salt thereof, is administered via an intravenous administration. 